Under Aim 1 of this project we have conducted necessary preclinical work for the initiation of a clinical trial testing CD22-targeted CAR T cells as a therapy for relapsed or resistant pre-B ALL. CD22 is expressed on 95 % of pre-B cell ALL and there is extensive expertise in the Pediatric Oncology Branch in CD22-targeted therapy for ALL using immunotoxin conjugated antibodies. This trial will utilize lentiviral-based viral vectors for genetic modification of patient-derived expanded T cells to be reinfused after lymphodepleting chemotherapy. The study design will be a phase I cell dose escalation study with an expansion cohort where an approximately 30% complete remission rate will used to determine whether this construct will be developed further. First, a number of important finding resulted from the pre-clinical work that have substantially streamlined the process of generated CAR T cells. Second, this trial will be critical in establishing whether the success of CD19 CAR T cell therapy can be extended to other targets on ALL. Third, since CD19 negative relapse has been observed following CD19 CAR therapy, a second target will potentially increase the overall curative potential of CAR therapy for ALL. This trial is undergoing the regulatory review process and is anticipated to open by the fourth quarter of 2014 and is expected to be completed in 1.5 years. We are also performing necessary pre-clinical work for the incorporation of an alternative, semi-automated T cell expansion and transduction that will further streamline the production process. Under Aim 2 of this project we have developed a number of novel CATR constructs including a TSLPR-targeted CAR for which we hold a patent. In addition, we have generated a CD19/CD22 bispecific CAR with excellent in vivo activity. This construct can prevent the emergence of target-loss variants of ALL in an in vitro system. Interestingly, using this in vitro system as well as in vivo xenograft system we are studying the mechanisms of antigen loss following CAR therapy. Second, we are developing novel acute myelogenous leukemia CAR constructs that may reduce the on-target off-tumor toxicity associated with myeloid antigens. Finally, we are developing additional CAR constructs for a very high-risk subset of T cell ALL (Early Thymic Precursor or ETP ALL). Under the third Aim of this project we are utilizing a murine CD19 CAR obtained form Dr. James Kochenderfer in conjunction with a transplantable ALL syngeneic model developed in our laboratory (see Project ZIA BC 011295) to study immunobiological principles associated with CAR therapy including the optimization CAR persistence and the mechanism for severe cytokine storm that can be seen in patients receiving CAR T cells. This model is also providing insights into antigen loss following CAR T cell therapy. Importantly, all of these questions cannot be addressed in the xenograft systems that require highly immunodeficient mice. Work conducted under ZIA BC 011295 has demonstrated the immunosuppressive effects of ALL on T cells. Aim 3 of this project will test whether redirection of T cell specificity using CAR constructs will overcome these immunosuppressive effects and whether blockade of negative regulators of the immune system can improve on the efficacy of CAR T cells.